The incidence of obesity has increased dramatically over the last fifty years. Over half of all Americans are over-weight and over one third are classified as clinically obese. The health ramifications of this epidemic of obesity are seen in th concomitant increase in the incidence of type 2 diabetes. An estimated 25 million Americans have diabetes with 95% of those being type 2. The most commonly prescribed therapeutic drugs used for type 2 diabetes target the AMP-activated protein kinase (AMPK). Therefore, understanding how AMPK is normally regulated is of great significance to human health. This proposal uses baker's yeast as its model system to study the regulation of the yeast AMPK. In both yeast and human cells, the overall structure of AMPK as well as its modes of regulation is highly conserved. The speed and synergy of genetic and biochemical studies in yeast make this an ideal system to dissect the regulation of AMPK. Past studies from our lab have shown that the activation of Snf1, the yeast name for AMPK, involves at least two steps: phosphorylation of the Snf1 activation loop and a second activation step mediated by the beta and gamma regulatory subunits of the Snf1 complex. We have shown that the first step, control of activation loop phosphorylation, is not regulated at the level of phosphate addition. Surprisingly, it is the dephosphorylation step that responds to cellular energy status. In recent studies we have been able to recapitulate the regulated dephosphorylation of Snf1 kinase in vitro in a purified system. Under conditions of energy stress, Snf1 is primarily in the active phosphorylated state. We can show that binding to low energy adenylate ligands promotes the formation of a phosphatase resistant conformation, thus stabilizing the active form of Snf1. Specific aim 1 of this proposal will characterize the molecular mechanism by which adenylate ligands promote the formation of the phosphatase resistant state using purified Snf1 enzymes. In other studies, we have shown that the ligand-mediated protection from dephosphorylation is only one level of regulation. Additional means of regulation must also be operating. In Specific aim 2, we will use genetic and biochemical screens to identify additional components and signaling pathways that regulate Snf1 kinase activity in vivo. In the final aim we examine the mechanism by which the phosphatase which inactivates Snf1 is targeted to specific substrates. In all, these studies will further our understanding of the regulation of AMPK and will help to develop new and more effective interventions to treat diabetes.